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Experimental Parasitology 167 (2016) 61e66

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Experimental Parasitology

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Phylogenetic position of Leishmania isolates from KhyberPakhtunkhwa province of Pakistan

Nazma Habib Khan a, c, *, Louisa A. Messenger b, Sobia Wahid a, c, Colin J. Sutherland a

a Department of Immunology & Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, WC1E 7HT, UKb Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, WC1E 7HT, UKc Department of Zoology, University of Peshawar, Peshawar, Khyber Pakhtunkhwa, Pakistan

h i g h l i g h t s

* Corresponding author. Department of Zoology, Uwar, Khyber Pakhtunkhwa, Pakistan.

E-mail addresses: nazma@upesh.edu.pk (N.H. Khaac.uk (L.A. Messenger), sobiawahid@upesh.edu.pk (Slshtm.ac.uk (C.J. Sutherland).

http://dx.doi.org/10.1016/j.exppara.2016.05.0060014-4894/© 2016 The Author(s). Published by Elsev

g r a p h i c a l a b s t r a c t

� Phylogenetic placement of Leish-mania spp. from Khyber Pak-htunkhwa (KP), Pakistan.

� Leishmania tropica complex differen-tiated into cytb genotypes A & B; 5subgenotypes in KP.

� KP Leishmania infantum affinity tovisceralizing Leishmania donovani &other cutaneous L. infantum.

� Phylogenetic affinities in KP’s spp. areuseful in tracing possible importroutes.

a r t i c l e i n f o

Article history:Received 8 April 2016Received in revised form20 May 2016Accepted 21 May 2016Available online 24 May 2016

Keywords:LeishmaniaCytochrome b (cytb)Internal transcribed spacer 2 (its2)Khyber Pakhtunkhwa (KP)

a b s t r a c t

Several species of the genus Leishmania are causative agents of cutaneous leishmaniasis in Pakistan. Thisstudy aimed to determine phylogenetic placement of Leishmania species causing cutaneous leishmani-asis in Khyber Pakhtunkhwa province, Pakistan (34 Leishmania tropica, 3 Leishmania infantum), in-relation to species from other geographical areas using gene sequences encoding cytochrome b (cytb)and internal transcribed spacer 2 (its2). Based on cytochrome b sequence analysis, L. tropica strains fromPakistan and other geographical regions were differentiated into two genotype groups, A and B. Withinthe province, five distinct L. tropica genotypes were recognized; two in group A, three in group B. Two L.infantum isolates from the province were closely associated with both Afro-Eurasian and Americanspecies of the Leishmania donovani complex, including Leishmania chagasi, L. infantum and L. donovanifrom Sudan and Ethiopia; while a third L. infantum isolate could not be differentiated from visceralizingKenyan and Indian L. donovani. We observed apposite phylogenetic placement of CL-causing L. tropicaand L. infantum from Khyber Pakhtunkhwa. Affinities ascribed to Leishmania spp. From the region arevaluable in tracing potential importation of leishmaniasis.© 2016 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license

(http://creativecommons.org/licenses/by/4.0/).

niversity of Peshawar, Pesha-

n), louisa.messenger@lshtm.. Wahid), colin.sutherland@

ier Inc. This is an open access artic

1. Introduction

Leishmaniasis, caused by protozoan trypanosomatid parasites ofgenus Leishmania, affects skin (cutaneous), mucous membranes(mucocutaneous) or internal organs (visceral), primarily inhumans. Recent figures suggest an approximate incidence of0.2e0.4 million visceral leishmaniasis (VL) and 0.7 to 1.2 million

le under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

N.H. Khan et al. / Experimental Parasitology 167 (2016) 61e6662

cutaneous leishmaniasis (CL) cases per annum. Compared to VL, CLis morewidely distributed; about one third of cases occur in each ofthree regions, the Americas, the Mediterranean basin and westernAsia through Middle East to Central Asia (Alvar et al., 2012).

Taxonomy of Leishmania has been contentious since the genuswas first described (Leishman, 1903). Lainson and Shaw (1987)classified Leishmania into sub-genera (L.) Leishmania, (L.) Vianniaand (L.) Sauroleishmania on the basis of parasite developmentwithin the sandfly gut and by differences in extrinsic characterssuch as geographical distribution, vector preferences, clinical out-comes (VL, CL or MCL) and antigenic characteristics (Lainson andShaw, 1987; Pratt and David, 1981). To date, multilocus enzymeelectrophoresis (MLEE) remains the recognized gold standard forLeishmania species identification (Rioux et al., 1990). However, thistraditional classification has been challenged using nuclear andmitochondrial molecular markers (Kuhls et al., 2007; Mauricioet al., 2004, 2006; Zemanova et al., 2004). There are a number ofputative Leishmania species for whom species status is still underquestion including Leishmania panamensis, Leishmania peruvianaand Leishmania lainsoni of sub-genus (L.) Viannia and L. garnhami,Leishmania pifanoi, Leishmania infantum, Leishmania chagasi, Leish-mania killicki and Leishmania archibaldi of sub-genus (L.) Leishmania(Banuls et al., 2007).

Phylogenetic relationships in Leishmania have previously beenresolved either within sample sets spanning the entire genus, oramong species only from Afro-Eurasia, the Americas, or a particulargeographic region, utilizing diverse loci including, ribosomal DNA(internal transcribed spacer (its) 1 and 2) (Berzunza-Cruz et al.,2002; Cupolillo et al., 1995; Da

vila and Memen, 2000; El Tai et al.,2000; Kuhls et al., 2005; Parvizi et al., 2008; Schonian et al.,2000; Spanakos et al., 2008; Sukmee et al., 2008; Villinski et al.,2008; Yang et al., 2010), DNA and RNA polymerase genes (Croanand Ellis, 1996; Croan et al., 1997), glycoprotein 63 gene (gp63)(Mauricio et al., 2007), cytochrome oxidase II (Ibrahim and Barker,2001), cytochrome b (cytb) (Asato et al., 2009; Luyo-Acero et al.,2004), mini-exon (Sukmee et al., 2008), 7SL RNA (Zelazny et al.,2005), hsp70 (Fraga et al., 2010) and repetitive DNA sequences(Piarroux et al., 1995).

Such phylogenetic studies are highly relevant in the context ofdisease control, since identifying discrete genetic groups canimprove our understanding of virulence, transmissibility and drugsusceptibility. In this study, cytochrome b (cytb) and internallytranscribed region 2 (its2) were targeted to investigate the phylo-genetic affinities of Leishmania tropica and L. infantum samples fromKhyber Pakhtunkhwa (KP), Pakistan in-relation to species fromother geographical areas.

2. Materials and methods

2.1. Leishmania isolates and DNA extraction

Parasite isolates were collected from patients suspected of CL(N ¼ 125) visiting Dermatology outpatient units in three majorhospitals of Peshawar, Khyber Pakhtunkhwa (KP), Pakistan in 2010.The Research Ethics Committee of the London School of Hygieneand Tropical Medicine (reference number 5677), UK and the Ethicscommittee at the Department of Pharmacy, University of Peshawar,Khyber Pakhtunkhwa, Pakistan (ref. #28/Pharm) approved thestudy. Details of the clinical protocol are given elsewhere (Khanet al., 2016 in prep). To mention briefly, localized lesions fromthese patients were sanitized and punctured with sterile lancets.Exudates collected using sterile pastettes were dispensed intobiphasic culture medium containing rabbit blood agar and M199medium (Sigma) supplemented with 10% heat inactivated fetal calfserum (HIFCS). DNA was extracted from pellets of positive cultures

using commercial QIAGEN DNeasy Blood and Tissue Kit. For somepatients DNAwas also extracted from filter papers (coarse porosity)with impressions from lesions or their biopsies using resin-basedChelex® method (Plowe et al., 1995). All samples (cultures and fil-ter papers) were pre-identified to Leishmania species level (usingKDNA primers) (Noyes et al., 1998) before they were subjected tocytb or its2 amplification.

In addition to the cultures (N ¼ 34) and filter papers (N ¼ 111)acquired from fieldwork in KP, DNAwas also extracted from in vitrocultures of L. tropica (N ¼ 88) and Leishmania donovani (N ¼ 2)strains belonging to other geographical regions from the cryobankrepository at the LSHTM (see Supplementary Table S1 for detail).

For inter-species phylogenetic analysis, a number of cytb(N ¼ 41) and its2 (N ¼ 58) sequences were retrieved from GenBank(Supplementary Table S1 and S2).

2.2. Cytb amplification and sequencing

Cytb, a maxicircle gene, was amplified using nested primersdescribed by Luyo-Acero et al. (2004) Nest1 (N1) primers corre-sponded to COIII (cytochrome c oxidase subunit III) and MURF4(maxi-circle unidentified reading frame 4). Nest2 (N2) primersLCBF1 and LCBR2 amplified approximately 866 bp of the internalcytb region. The cycling conditions for N1 and N2 included, aninitial incubation for 1 min at 94 �C, followed by 40 cycles of 94 �Cfor 1 min, 50 �C for 1 min, 72 �C for 1 min and a final extension for5 min at 74 �C. DNAwas extracted from gel sections using QIAquickGel extraction Kit (QIAGEN). DNA sequencing was carried out usinga set of internal primers LCBF4 and LCBR4 followingmanufacturer’sprotocol for BigDye™ terminator cycle sequencing kit V3.1.

2.3. its2 amplification and sequencing

The its2 region of chromosome number 27 was amplified (Bulleet al., 2002). Cycling conditions employed had an initial incubationfor 2 min at 94 �C, followed by 40 cycles of 94 �C for 20 s, 53 �C for30 s, 72 �C for 1 min and a final extension of 72 �C for 10 min.Amplicons excised from gel were processed by QIAquick Gelextraction Kit (QIAGEN). Extracted DNA fragments were sequencedusing BigDye™ sequencing kit V3.1. Three L. tropica strains (MRAT/IQ/73/ADHANIS, MHOM/AZ/1974/SAF-K27 and MHOM/PK/1987/FAZAL) were sequenced from clones derived using the CloneJET™PCR cloning kit (Fermentas). We specifically undertook PCR cloningof multiple sequences per parasite isolate to circumvent the prob-lems of differing lengths of variable regions.

2.4. Cytb and its2 phylogenetic analysis

Sequence chromatograms were analyzed and edited in Gene-ious v5.5.7 (Kearse et al., 2012). These sequences along with thoseretrieved from GenBank were used to derive Maximum likelihood(ML) trees and to perform, in parallel, Bayesian analysis. MEGAv5.05 (Tamura et al., 2011) was used to align sequences and toidentify single nucleotide polymorphisms (SNPs). Trypanosomacruzi and Herpetomonas muscarum were treated as out-groups incytb and its2 analyses respectively. Alignments created from MEGAv5.05 were run in jMODELTEST v1.0 to select a best fit nucleotidesubstitution model (Posada, 2008).

For cytb the best model selected for the dataset was used toconstruct a Maximum Likelihood tree (ML) tree in MEGA V5.05.Bayesian analysis using the same model was carried out in TOPALiv2.5 (Milne et al., 2004) to produce posterior probabilities (abbre-viated as PP) supporting the topology of ML bootstrap (abbreviatedas BP) tree. For its2, both ML tree and Bayesian phylogenetic anal-ysis was carried out in TOPALi v2.5 under same settings used for

N.H. Khan et al. / Experimental Parasitology 167 (2016) 61e66 63

cytb. Cytb and its2 targets sequenced from this study have beensubmitted to GenBank (KT972143-KT972260, KT972262-KT972277).

3. Results and discussion

Using the diagnostic nested PCR of Noyes et al. (1998), all cul-tures acquired from KP were identified as L. tropica (N ¼ 34).Furthermore three L. infantum (N ¼ 3) were identified from filterpaper spots, although they could not be successfully established incultures.

Cytb genotypes from a total of 122 L. tropica cultures wereanalyzed along with an additional set of 41 sequences retrievedfrom the GenBank. No cytb product could be amplified from filterpaper samples identified as L. infantum from Pakistan and thuscould not be included in the analysis (Supplementary Table S1).Approximately 653 bp (with gaps) cytb gene alignment wasanalyzed.

Interpretable sequence data for its2 could be obtained from onlya single L. tropica and three L. infantum from KP, Pakistan. Ampli-cons from each of three previously archived L. tropica isolatesnamely MRAT/IQ/73/ADHANIS, MHOM/AZ/1974/SAF-K27 andMHOM/PK/1987/FAZAL produced two ITS2 haplotypes (H1 andH2), suggesting heterozygosity. An additional 58 its2 sequences

Fig. 1. Cytb maximum-likelihood consensus tree constructed from the Leishmania sppcomplex samples typed in this study are expressed as genotypes (Supplementary Table S1Consensus tree was constructed from 1000 bootstrap replicates of cytb sequences along witsimulations with 25% burn-in period) using GTR þ G þ I (General time reversible model wmodel. Only significant bootstraps (�50%) and posterior probabilities (�0.50) are shown in

were retrieved from GenBank (Supplementary Table S2). Approxi-mately, 656 bp (with gaps) its2 alignment was analyzed.

3.1. Phylogenetic position of Pakistani L. tropica

Amongst all the L. tropica cytb sequences (122 from the studyand 2 from Genbank) analyzed in the present study, 39 variablesites were identified in the sequence alignment giving a total of 21genotypes. The co-occurrence of 15 single nucleotide poly-morphisms (SNPs) within the L. tropica dataset discriminated thesamples into 2 groups, A (12 genotypes among 101 strains) and B (9genotypes among 22 strains). The most common genotype in thedataset was a genotype of group A (A1) observed among 91 of 124 L.tropica strains. Within KP province of Pakistan, 5 genotypes wereobserved (2 genotypes in group A and 3 genotypes in group B)(Supplementary Table S1). L. tropica species complex was sup-ported bymoderate bootstraps in the cytbML tree (BP¼ 60) (Fig. 1).

A moderately-supported L. tropica cluster (BP ¼ 95; PP ¼ 0.99),consisting of all its haplotypes, was observed in the its2 ML tree(Fig. 2). Within L. tropica, there were sub-clusters of clones withboth haplotypes (H1 and H2) of its2 (including ones from the cur-rent study), although not supported by significant bootstraps (i.e.>50%). The single successfully sequenced L. tropica isolate fromNorthern Pakistan sampled in the present study (MHOM/PK/2010/

. typed in the current study and those retrieved from Genbank. Data for L. tropica). Strains with no species designations are expressed with standard strain codes only.h Bayesian analysis (5 independent runs over 1 million generations sampling every 10ith gamma distributed rates and invariant sites) as the best-fit nucleotide substitutionred. *Outgroup.

Fig. 2. its2 maximum-likelihood consensus tree constructed from the Leishmania spp. typed in the current study and those retrieved from Genbank. Strains with no speciesdesignations are expressed with standard strain codes only. The consensus tree was constructed from 1000 bootstrap replicates of its2 sequences (656 bp). Consensus tree wasconstructed from 1000 bootstrap replicates of its2 sequences along with Bayesian analysis (5 independent runs over 1 million generations sampling every 10 simulations with 25%burn-in period) using SYM þ G (Symmetric model with gamma distributed rate variation among sites) as the best-fit nucleotide substitution model. Only significant bootstraps(�50%) and posterior probabilities (�0.50) are shown in red. *Outgroup.

N.H. Khan et al. / Experimental Parasitology 167 (2016) 61e6664

CMH061) was placed in the sub-cluster of H2 haplotype. Overall,homogeneity was observed among the L. tropica strains fromdifferent geographical sites, including the genetically discreteNamibian strain IROS/NA/76/ROSSI-II (Schonian et al., 2001;Schwenkenbecher et al., 2006) (Fig. 2).

In this study both the phylogenetic analyses (cytb and its2)placed L. tropica as a discrete species complex within the genus,comprising L. tropica and Leishmania aethiopica as its two inte-grated species (Figs. 1 and 2). Using hsp70, Fraga et al. (2010),however, could not assign an individual species status to L.aethiopica. L. killicki from Tunisia, given full species rank by MLEE(Rioux et al., 1990), is indistinguishable from L. tropica by bothphylogenetic topologies in the present study, a finding supportedby others (Schonian et al., 2001; Schwenkenbecher et al., 2006).The cytb tree generated in the present study identified two maingenotypes of L. tropica species, whereas its2 haplotypes werealmost homogenous throughout the L. tropica cluster. Evidently,its2 may not be a discriminatory target for resolution of intra-

specific diversity within L. tropica. This lack of congruence be-tween our two genotypic phylogenies at species level for L. tropicamay also be due to intrinsic differences in mutation rate betweenmitochondrial DNA as compared to nuclear DNA or evidence ofhistorical genetic recombination (Bastos-Silveira et al., 2012; Chenet al., 2009; Gomez-Zurita and Vogler, 2003; Messenger et al., 2012;Tibayrenc, 1999).

3.2. Phylogenetic position of Pakistani L. infantum

In the its2 analysis two of the L. infantum strains from Pakistan(MHOM/PK/2010/CMH041 and MHOM/PK/2010/CMH069) wereindistinctly placed within the L. donovani complex clade (BP ¼ 100;PP ¼ 1.00). A distinct sub-cluster in the main complex consisting ofSouth Asian (India, Sri Lanka and Bhutan) and Kenyan L. donovanistrains (BP ¼ 60; PP ¼ 0.97) also included a Pakistani L. infantumstrain MHOM/PK/2010/CMH052 from the present study (Fig. 2).Several reports have found Indian and Kenyan L. donovani are

N.H. Khan et al. / Experimental Parasitology 167 (2016) 61e66 65

genetically distinct from visceralizing agents in Sudan and Ethiopia(Ibrahim and Barker, 2001; Kuhls et al., 2005; Lewin et al., 2002;Lukes et al., 2007; Mauricio et al., 2001). Allocation of a presentlygenotyped Pakistani strain MHOM/PK/2010/CMH052 in this sub-cluster may either be a consequence of true similarity, or lack ofdiscrimination between sequences analyzed. However, this doesnot seem to be the case with the two other Pakistani isolates typedas L. infantum (MHOM/PK/2010/CMH041 and MHOM/PK/2010/CMH069), which were plausibly grouped with L. Infantum in the L.donovani complex cluster. Neither L. donovani nor CL infectionscaused by L. infantum have been previously reported in Pakistan.The three samples genotyped as L. infantum by KDNA from CL pa-tients in the present study are the first reports of such cases (Khanet al., 2016 in prep).

L. chagasi (L. infantum from the Americas) could not be differ-entiated from the Mediterranean, Chinese and Pakistani L. infantumspecies. This is in agreement with previous studies based on MLEE,RAPD and RFLP analysis of nuclear targets (its, gp63, microsatellitesand other protein coding genes) (Cupolillo et al., 1994; Kuhls et al.,2005; Lukes et al., 2007; Mauricio et al., 2001, 1999, 2000;Ochsenreither et al., 2006). Evidently both cytb and its2 phylog-enies produced herein supported a unitary status for the fourdefined species; L. donovani, L. archibaldi, L. infantum and L. chagasiin the L. donovani species complex (Figs. 1 and 2). Tibayrenc (2006)put forward a phylogenetic species concept that states, “A species isa monophyletic group composed of the smallest diagnosable clus-ter of individual organisms, withinwhich there is a parental patternof ancestry and descent”. Based on the demarcations of this conceptall the four named species in the L. donovani complex should bedefined together as a singular species. Several studies support thisnotion of full species status (Croan et al., 1997; Lukes et al., 2007;Luyo-Acero et al., 2004; Mauricio et al., 2000; Tibayrenc, 2006).

4. Conclusion

Phylogenetic topologies based on cytb and its2 placed CL-causing L. tropica and L. infantum from KP, Pakistan confidentlywithin their generally accepted respective species complexes; thelatter is the first report of cutaneous disease by L. infantum inPakistan. These observations highlight the need for further epide-miological study of parasites from the L. donovani complex inPakistan and its neighboring countries, to further clarify the taxo-nomic status of strains causing cutaneous and visceral leishmani-asis in Pakistan.

Acknowledgments

We thank Dr. Vanessa Yardley and Dr. Debbie Nolder for offeringaccess to the LSHTM cryo-repository. We are grateful to BrighidO’Neill and Dr. Vanessa Yardley for their efforts in establishingcultures from field collection in Pakistan. We would also like toacknowledge Dr. Arfan ul Bari and Dr. Rizwan Hashim (Departmentof Dermatology, Combined Military Hospital, Peshawar) for thevaluable assistance during fieldwork. This work was supported by“Faculty Development Program (FDP)” scholarship awarded byHigher Education Commission, Pakistan to NK.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.exppara.2016.05.006.

References

Alvar, J., Velez, I.D., Bern, C., Herrero, M., Desjeux, P., Cano, J., Jannin, J., den Boer, M.,

2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS ONE7, e35671.

Asato, Y., Oshiro, M., Myint, C.K., Yamamoto, Y.-i., Kato, H., Marco, J.D., Mimori, T.,Gomez, E.A.L., Hashiguchi, Y., Uezato, H., 2009. Phylogenic analysis of the genusLeishmania by cytochrome b gene sequencing. Exp. Parasitol. 121, 352e361.

Banuls, A.L., Hide, M., Prugnolle, F., 2007. Leishmania and the leishmaniases: aparasite genetic update and advances in taxonomy, epidemiology and patho-genicity in humans. Adv. Parasitol. 64, 1e109.

Bastos-Silveira, C., Santos, S.M., Monarca, R., Mathias Mda, L., Heckel, G., 2012. Deepmitochondrial introgression and hybridization among ecologically divergentvole species. Mol. Ecol. 21, 5309e5323.

Berzunza-Cruz, M., Cabrera, N., Crippa-Rossi, M., Sosa Cabrera, T., Perez-Montfort, R., Becker, I., 2002. Polymorphism analysis of the internal transcribedspacer and small subunit of ribosomal RNA genes of Leishmania mexicana.Parasitol. Res. 88, 918e925.

Bulle, B., Millon, L., Bart, J.-M., Gallego, M., Gambarelli, F., Portus, M., Schnur, L.,Jaffe, C.L., Fernandez-Barredo, S., Alunda, J.M., Piarroux, R., 2002. Practicalapproach for typing strains of Leishmania infantum by microsatellite analysis.J. Clin. Microbiol. 40, 3391e3397.

Chen, W., Bi, K., Fu, J., 2009. Frequent mitochondrial gene introgression among highelevation Tibetan megophryid frogs revealed by conflicting gene genealogies.Mol. Ecol. 18, 2856e2876.

Croan, D., Ellis, J., 1996. Phylogenetic relationships between Leishmania, Vianniaand Sauroleishmania inferred from comparison of a variable domain within theRNA polymerase II largest subunit gene. Mol. Biochem. Parasitol. 79, 97e102.

Croan, D.G., Morrison, D.A., Ellis, J.T., 1997. Evolution of the genus Leishmaniarevealed by comparison of DNA and RNA polymerase gene sequences. Mol.Biochem. Parasitol. 89, 149e159.

Cupolillo, E., Grimaldi Jr., G., Momen, H., 1994. A general classification of NewWorldLeishmania using numerical zymotaxonomy. Am. J. Trop. Med. Hyg. 50,296e311.

Cupolillo, E., Grimaldi Junior, G., Momen, H., Beverley, S.M., 1995. Intergenic regiontyping (IRT): a rapid molecular approach to the characterization and evolutionof Leishmania. Mol. Biochem. Parasitol. 73, 145e155.

Da

vila, A.M.R., Memen, H., 2000. Internal-transcribed-spacer (ITS) sequences usedto explore phylogenetic relationships within Leishmania. Ann. Trop. Med. Par-asitol. 94, 651e654.

El Tai, N.O., Osman, O.F., El Fari, M., Presber, W., Schonian, G., 2000. Genetic het-erogeneity of ribosomal internal transcribed spacer in clinical samples ofLeishmania donovani spotted on filter paper as revealed by single-strandconformation polymorphisms and sequencing. Trans. R. Soc. Trop. Med. Hyg.94, 575e579.

Fraga, J., Montalvo, A.M., De Doncker, S., Dujardin, J.-C., Van der Auwera, G., 2010.Phylogeny of Leishmania species based on the heat-shock protein 70 gene.Infect. Genet. Evol. 10, 238e245.

Gomez-Zurita, J., Vogler, A.P., 2003. Incongruent nuclear and mitochondrial phy-logeographic patterns in the Timarcha goettingensis species complex (Cole-optera, Chrysomelidae). J. Evol. Biol. 16, 833e843.

Ibrahim, M.E., Barker, D.C., 2001. The origin and evolution of the Leishmaniadonovani complex as inferred from a mitochondrial cytochrome oxidase II genesequence. Infect. Genet. Evol. 1, 61e68.

Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S.,Cooper, A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Meintjes, P.,Drummond, A., 2012. Geneious basic: an integrated and extendable desktopsoftware platform for the organization and analysis of sequence data. Bioin-formatics 28, 1647e1649.

Khan, N.H., Bari, A.U., Hashim, R., Khan, I., Muneer, A., Shah, A., Wahid, S., Yardley, V.,O’Neil, B., Sutherland, C.J., 2016. Cutaneous leishmaniasis in Khyber Pak-htunkhwa Province of Pakistan; Clinical Diversity and Species-level Diagnosis inprep.

Kuhls, K., Keilonat, L., Ochsenreither, S., Schaar, M., Schweynoch, C., Presber, W.,Schonian, G., 2007. Multilocus microsatellite typing (MLMT) reveals geneticallyisolated populations between and within the main endemic regions of visceralleishmaniasis. Microbes Infect. 9, 334e343.

Kuhls, K., Mauricio, I.L., Pratlong, F., Presber, W., Schonian, G., 2005. Analysis of ri-bosomal DNA internal transcribed spacer sequences of the Leishmania dono-vani complex. Microbes Infect. 7, 1224e1234.

Lainson, R., Shaw, J.J., 1987. Evolution, Classification and Geographical Distribution.In: Peters, W., Killick-Kendrick, R. (Eds.), The Leishmaniasis in Biology andMedicine. Academic Press, London.

Leishman, W.B., 1903. On the possibility of the occurrence of trypanomiasis in India.Br. M. J. 1, 1252e1254.

Lewin, S., Schonian, G., El Tai, N., Oskam, L., Bastien, P., Presber, W., 2002. Straintyping in Leishmania donovani by using sequence-confirmed amplified regionanalysis. Int. J. Parasitol. 32, 1267e1276.

Lukes, J., Mauricio, I.L., Schonian, G., Dujardin, J.-C., Soteriadou, K., Dedet, J.-P.,Kuhls, K., Tintaya, K.W.Q., Jirku, M., Chocholova, E., Haralambous, C., Pratlong, F.,Obornik, M., Horak, A., Ayala, F.J., Miles, M.A., 2007. Evolutionary andgeographical history of the Leishmania donovani complex with a revision ofcurrent taxonomy. Proc. Natl. Acad. Sci. U. S. A. 104, 9375e9380.

Luyo-Acero, G.E., Uezato, H., Oshiro, M., Takei, K., Kariya, K., Katakura, K., Gomez-Landires, E., Hashiguchi, Y., Nonaka, S., 2004. Sequence variation of the cyto-chrome b gene of various human infecting members of the genus Leishmaniaand their phylogeny. Parasitology 128, 483e491.

Mauricio, I.L., Gaunt, M.W., Stothard, J.R., Miles, M.A., 2001. Genetic typing and

N.H. Khan et al. / Experimental Parasitology 167 (2016) 61e6666

phylogeny of the Leishmania donovani complex by restriction analysis of PCRamplified gp63 intergenic regions. Parasitology 122, 393e403.

Mauricio, I.L., Gaunt, M.W., Stothard, J.R., Miles, M.A., 2007. Glycoprotein 63 (gp63)genes show gene conversion and reveal the evolution of Old World Leishmania.Int. J. Parasitol. 37, 565e576.

Mauricio, I.L., Howard, M.K., Stothard, J.R., Miles, M.A., 1999. Genomic diversity inthe Leishmania donovani complex. Parasitology 119 (Pt 3), 237e246.

Mauricio, I.L., Stothard, J.R., Miles, M.A., 2000. The strange case of Leishmaniachagasi. Parasitol. Today 16, 188e189.

Mauricio, I.L., Stothard, J.R., Miles, M.A., 2004. Leishmania donovani complex:genotyping with the ribosomal internal transcribed spacer and the mini-exon.Parasitology 128, 263e267.

Mauricio, I.L., Yeo, M., Baghaei, M., Doto, D., Pratlong, F., Zemanova, E., Dedet, J.P.,Lukes, J., Miles, M.A., 2006. Towards multilocus sequence typing of the Leish-mania donovani complex: resolving genotypes and haplotypes for five poly-morphic metabolic enzymes (ASAT, GPI, NH1, NH2, PGD). Int. J. Parasitol. 36,757e769.

Messenger, L.A., Llewellyn, M.S., Bhattacharyya, T., Franzen, O., Lewis, M.D.,Ramirez, J.D., Carrasco, H.J., Andersson, B., Miles, M.A., 2012. Multiple mito-chondrial introgression events and heteroplasmy in trypanosoma cruzirevealed by maxicircle MLST and next generation sequencing. PLoS Negl. Trop.Dis. 6, e1584.

Milne, I., Wright, F., Rowe, G., Marshall, D.F., Husmeier, D., McGuire, G., 2004.TOPALi: software for automatic identification of recombinant sequences withinDNA multiple alignments. Bioinformatics 20, 1806e1807.

Noyes, H.A., Reyburn, H., Bailey, J.W., Smith, D., 1998. A nested-PCR-based schizo-deme method for identifying Leishmania kinetoplast minicircle classes directlyfrom clinical samples and its application to the study of the epidemiology ofLeishmania tropica in Pakistan. J. Clin. Microbiol. 36, 2877e2881.

Ochsenreither, S., Kuhls, K., Schaar, M., Presber, W., Schonian, G., 2006. Multilocusmicrosatellite typing as a new tool for discrimination of Leishmania infantumMON-1 strains. J. Clin. Microbiol. 44, 495e503.

Parvizi, P., Moradi, G., Akbari, G., Farahmand, M., Ready, P.D., Piazak, N., Assmar, M.,Amirkhani, A., 2008. PCR detection and sequencing of parasite ITS-rDNA genefrom reservoirs host of zoonotic cutaneous leishmaniasis in central Iran. Para-sitol. Res. 103, 1273e1278.

Piarroux, R., Fontes, M., Perasso, R., Gambarelli, F., Joblet, C., Dumon, H., Quilici, M.,1995. Phylogenetic relationships between Old World Leishmania strainsrevealed by analysis of a repetitive DNA sequence. Mol. Biochem. Parasitol. 73,249e252.

Plowe, C.V., Djimde, A., Bouare, M., Doumbo, O., Wellems, T.E., 1995. Pyrimethamineand proguanil resistance-conferring mutations in Plasmodium falciparumdihydrofolate reductase: polymerase chain reaction methods for surveillance inAfrica. Am. J. Trop. Med. Hyg. 52, 565e568.

Posada, D., 2008. jModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25,1253e1256.

Pratt, D.M., David, J.R., 1981. Monoclonal antibodies that distinguish between newworld species of Leishmania. Nature 291, 581e583.

Rioux, J.A., Lanotte, G., Serres, E., Pratlong, F., Bastien, P., Perieres, J., 1990. Taxonomyof Leishmania. Use of isoenzymes. Suggestions for a new classification. Ann.Parasitol. Hum. Comp. 65, 111e125.

Schonian, G., Akuffo, H., Lewin, S., Maasho, K., Nylen, S., Pratlong, F.,Eisenberger, C.L., Schnur, L.F., Presber, W., 2000. Genetic variability within thespecies Leishmania aethiopica does not correlate with clinical variations ofcutaneous leishmaniasis. Mol. Biochem. Parasitol. 106, 239e248.

Schonian, G., Schnur, L., el Fari, M., Oskam, L., Kolesnikov, A.A., Sokolowska-Kohler, W., Presber, W., 2001. Genetic heterogeneity in the species Leishmaniatropica revealed by different PCR-based methods. Trans. R Soc. Trop. Med. Hyg.95, 217e224.

Schwenkenbecher, J.M., Wirth, T., Schnur, L.F., Jaffe, C.L., Schallig, H., Al-Jawabreh, A.,Hamarsheh, O., Azmi, K., Pratlong, F., Schonian, G., 2006. Microsatellite analysisreveals genetic structure of Leishmania tropica. Int. J. Parasitol. 36, 237e246.

Spanakos, G., Piperaki, E.T., Menounos, P.G., Tegos, N., Flemetakis, A., Vakalis, N.C.,2008. Detection and species identification of Old World Leishmania in clinicalsamples using a PCR-based method. Trans. R. Soc. Trop. Med. Hyg. 102, 46e53.

Sukmee, T., Sriripattanapipong, S., Mungthin, M., Worapong, J., Rangsin, R.,Samung, Y., Kongkaew, W., Bumrungsana, K., Chanachai, K., Apiwathanasorn, C.,Rujir- ojindakul, P., Wattanasri, S., Ungchusak, K., Leelayoova, S., 2008.A suspected new species of Leishmania, the causative agent of visceral leish-maniasis in a Thai patient. Int. J. Parasitol. 38, 617e622.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5:molecular evolutionary genetics analysis using maximum likelihood, evolu-tionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28,2731e2739.

Tibayrenc, M., 1999. Toward an integrated genetic epidemiology of parasitic pro-tozoa and other pathogens. Annu. Rev. Genet. 33, 449e477.

Tibayrenc, M., 2006. The species concept in parasites and other pathogens: apragmatic approach? Trends Parasitol. 22, 66e70.

Villinski, J.T., Klena, J.D., Abbassy, M., Hoel, D.F., Puplampu, N., Mechta, S., Boakye, D.,Raczniak, G., 2008. Evidence for a new species of Leishmania associated withfocal disease outbreak in Ghana. Diagn. Microbiol. Infect. Dis. 60.

Yang, B.B., Guo, X.G., Hu, X.S., Zhang, J.G., Liao, L., Chen, D.L., Chen, J.P., 2010. Speciesdiscrimination and phylogenetic inference of 17 Chinese Leishmania isolatesbased on internal transcribed spacer 1 (ITS1) sequences. Parasitol. Res. 107,1049e1065.

Zelazny, A.M., Fedorko, D.P., Li, L., Neva, F.A., Fischer, S.H., 2005. Evaluation of 7SLRNA gene sequences for the identification of Leishmania spp. Am. J. Trop. Med.Hyg. 72, 415e420.

Zemanova, E., Jirku, M., Mauricio, I.L., Miles, M.A., Lukes, J., 2004. Genetic poly-morphism within the leishmania donovani complex: correlation withgeographic origin. Am. J. Trop. Med. Hyg. 70, 613e617.